The relation between polyoma T-antigen and increased 5S RNA synthesis in cell-free extracts from polyoma-infected mouse kidney cell cultures

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Article

Reference

The relation between polyoma T-antigen and increased 5S RNA synthesis in cell-free extracts from polyoma-infected mouse kidney

cell cultures

MATTER, Jean-Marc, WEIL, Roger

Abstract

In polyoma-infected mouse kidney cell cultures 5S RNA synthesis began to increase around 16 h, i.e. 7–9 h after the onset of polyoraa T-antigen synthesis. The rate of polyoma-induced 5S RNA synthesis reached a maximum plateau around 25 h when it was 1.8–2.0 times higher than in mock-infected parallel cultures. Stimulation of 5S RNA synthesis in vivo thus coincided in time with the increase in total cellular RNA and protein. Cell-free extracts (S100) prepared at 15 h from mock-(S100-M) or polyoma-infected (S100-Py) mouse kidney cell cultures were indistinguishable with respect to protein concentration and 5S RNA synthesis, using a cloned somatic Xenopus borealis 5S gene as template. S100-Py extracted 25 h after infection contained 30% more protein and synthesized 1.5–2.0 times more 5S RNA than S100-M.

Complete removal of the polyoma T-antigens from S100-Py by 3 cycles of immunoprecipitation with hamster anti-T serum remained without effect on stimulated 5S RNA synthesis. However, a linear relationship between 5S RNA synthesis and protein concentration of S100-M and S100-Py was observed.

MATTER, Jean-Marc, WEIL, Roger. The relation between polyoma T-antigen and increased 5S RNA synthesis in cell-free extracts from polyoma-infected mouse kidney cell cultures. Nucleic Acids Research , 1982, vol. 10, no. 23, p. 7643-7655

DOI : 10.1093/nar/10.23.7643

Available at:

http://archive-ouverte.unige.ch/unige:150093

Disclaimer: layout of this document may differ from the published version.

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The relation between potyoma T-antigen and increased 5S RNA synthesis in ceO-free extracts from polyoma-infected mouse kidney cell cultures

Jean-Marc Matter and Roger Weil

Department of Molecular Biology, University of Geneva, Geneva, Switzerland Received 13 September 1982; Accepted 2 November 1982

ABSTRACT

In polyoma-infected mouse kidney cell cultures 5S RNA synthesis began to increase around 16 h, i.e. 7-9 h after the onset of polyoraa T-antigen synthesis. The rate of

polyoma-induced 5S RNA synthesis reached a maximum plateau around 25 h when it was 1.8-2.0 times higher than in mock-infected parallel cultures. Stimulation of 5S RNA

synthesis in vivo thus coincided in time with the increase in total cellular RNA and protein. Cell-free extracts (S100) prepared at 15 h from mock-(S100-M) or polyoma-infected

(SlOO-Py) mouse kidney cell cultures were indistinguishable with respect to protein concentration and 5S RNA synthesis, using a cloned somatic Xenopus borealis 5S gene as template.

SlOO-Py extracted 25 h after infection contained 30% more protein and synthesized 1.5-2.0 times more 5S RNA than S100-M.

Complete removal of the polyoma T-antigens from SlOO-Py by 3 cycles of immunoprecipitation with hamster anti-T serum remained without effect on stimulated 5S RNA synthesis.

However, a linear relationship between 5S RNA synthesis and protein concentration of S100-M and SlOO-Py was observed.

INTRODUCTION

In quiescent mouse or monkey kidney tissue culture cells arrested in phase Go/Gl of the mitotic cycle synthesis of polyoma- or SV4O- T-antigens, respectively, is followed by a mitotic host response (1); it comprises stimulation of overall cellular RNA (hnRNA, rRNA, tRNA and 5S RNA) and protein

synthesis (2; Matter, J.-M., Khandjian, E.W. and Weil, R. , unpublished results), the activation of the cellular

DNA-synthesizing apparatus and the duplication of the host cell chromatin (S-phase). In lytic infection virus-induced S-phase is paralleled by the production of progeny virus and is followed by cell lysis. The early events of infection, including the activation of the DNA-synthesizing apparatus,

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occur with the same kinetics if DNA synthesis is inhibited with 5-fluorodeoxyuridine or cytosine arabinoside (3,4). The close temporal relationship between the appearance of T-antigens and the stimulation of cellular RNA synthesis pointed to a

causative relationship (1). Several lines of experimental observations led to the suggestion that SV40 T-antigens primarily and directly stimulated transcription of the ribosomal genes (5).

In the present work we measured polyoma-induced

stimulation of 5S RNA synthesis in primary mouse kidney cell cultures; from parallel cultures we prepared cell free-extracts

(S100) and studied the effect of the polyoma T-antigens on transcription in vitro of a cloned somatic 5S gene from Xenopus borealis (6,7).

MATERIALS AND METHODS

Cell cultures and virus infection.

Confluent primary mouse kidney cell cultures (8) containing about 10 cells per dish (90 mm diameter) were infected with wild type polyoma viral preparations containing

Q

5.10 plaque forming units per ml; after virus adsorption for 90 min the cultures were covered with reinforced Eagle's medium in the absence of added proteins (1). Parallel cultures were mock-infected with culture medium. In all experiments reported

in this paper, DNA synthesis was inhibited with 15 ug/ml 5-fluorodeoxyuridine (Hoffman-La Roche, Basel) present in the medium used to cover the cultures after virus adsorption.The cultures were kept at 37°C in a CO.,-incubator for the lengths of time indicated in Results.

Extraction of cytoplasmic RNA.

For each experimental point 4 mock- and 4 polyoma- infected mouse kidney cell cultures were used. At the times indicated in Results, the cultures were pulse-labeled for 1 h with 50 uCi [5-3H]uridine (20-25 Ci/mM, New England

Corporation). At the end of the pulse the cultures were washed twice with ice cold 10 ml TNM buffer (1O mM triethanolamine pH 7.4, 0.25 M sucrose, 25 mM NaCl, 5 mM MgCl2); the cells were lysed with 1 ml/ dish of TNM buffer containing 1% Nonidet-P40

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(Fluka); the lysates were collected, passed 10 times through a 21-gauge needle fitted to a 1 ml syringe and then centrifuged at 1500 x g at 4°C for 15 min. The supernatant is referred to as "cytoplasmic fraction" and the pellet as "nuclear fraction".

Cytoplasmic RNA was extracted with phenol-chloroform-isoamyl- alcohol (5O/50/1) (9) and nuclear RNA with hot phenol (10); RNA was precipitated from the aqueous phase with 2 volumes of ethanol and then analysed by electrophoresis in slab gels (see below) or in tubular polyacrylamide-agarose gels (11).

Preparation of cell-free extracts (S100) .

S100 from mock- and polyoma-infected cell cultures was prepared as described by Weil et al. (12) with minor modi-

fications. All procedures were performed at 4 C. At the times o

indicated in Results, 10 cultures (containing about 10 cells) were washed twice with 10 ml phosphate-buffered saline

(137 mM NaCl, 2.7 mM KC1, 8.0 mM Na2HPO4, 1.5 mM KH2PO4, 0.5 mM MgCl_, 0.9 mM C a d - ) and then with 10 ml transcription buffer (10 mM Hepes pH 7.9, 10 mM KC1, 1.5 mM MgCl2, 0.5 mM dithiotreitol). Ten min later the transcription buffer was removed from the cultures, the cells were scraped from the dishes with a silicone rubber, passed 10 times through a 21-gauge needle fitted to a 1 ml syringe and then centrifuged at 3000 x g for 10 min. The pellet contained morphologically intact nuclei, judged by phase contrast microscopy .The low speed supernatant was recentrifuged at 100'000 x g for 1 h and the resulting high speed supernatant (about 1 ml) is referred to as S100-M (mock-infected) or SlOO-Py (polyoma-infected).

Protein content of the S100 was determined according to Lowry (13). In some experiments (see Results) S100 was incubated with 100 jig/ml of DNAse I (Worthington, RNAse free) for 1 h at 37°C.

Radioimmune assay for polyoma T-antiqens.

Three polyoma- or mock-infected cultures each were labeled from 15 to 25 h after infection with 50 uCi [ S]methionine (50O-10OO Ci/mM, Radiochemical Centre, Amersham) and S100 0.3 ml) was prepared as described above.The nuclear fraction from the low speed centrifugation was resuspended in 0.3 ml NET buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.01% NaN-, 0.5% Nonidet-P40) and sonicated at 75 W for 30 s

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(MSE sonicator) (14) . For the radioimmune assay (15,16) 10 ul of polyoraa anti-T serum from tumor bearing hamsters, a gift from Dr Hans Ttlrler, was added to 1OO ul S100 or 100 ul sonicate, respectively. The mixtures were incubated at 4°C for 3 h. To aliguots of 100 ul S100 or 100 ul sonicate, we added 10 ul normal hamster serum or 10 (il rabbit antiserum directed against bovine serum albumin (BSA), a gift from Dr Timothy Rose. All samples were mixed with 200 ul protein A-sepharose and then incubated at 4 C for 1 h. Thereafter the antigen-antibody complexes bound to protein A-sepharose were pelleted by centrifugation at 1000 x g for 5 min. The supernatant was removed and subjected to a second and third cycle of immunoprecipitation.

The final pellet was washed 3 times with 5 ml NET buffer and twice with 5 ml NET buffer containing 2 M urea. The T-antigens were eluted from the protein A-sepharose with 50 ul sample buffer (65 mM Tris-HCl pH 6.8, 2% SDS, 2% mercaptoethanol, 15% glycerol, 0.01% bromophenol) and were then analysed by electrophoresis in a SDS/polyacrylamide (12.5% acrylamide) slab gel (17). The gels were fixed, stained with Coomassie brillant blue and then processed for fluorography (18).

In vitro transcription of 5S DNA.

The in vitro RNA synthesis system was that described by Weil et al.(12). The plasmid used (pMB9Xbsl) contained a single somatic ribosomal 5S gene from Xenopus borealis inserted in the vector pMB9. The plasmid was a gift from Dr Donald D. Brown

(Carnegie Institution of Washington) to Dr Marco Crippa (Department of Animal Biology, University of Geneva). The standard RNA synthesis system contained in a total volume of 50 ul, 600 uM of CTP, GTP and ATP each, 100 uM UTP, 10 mM Hepes pH 7.8, 50O mM dithiotreitol, 75 mM KC1, 2.5 mM MgCl-, 3 ug plasmid DNA and 10 uCi 5'[o32P]UTP (400-600 Ci/mM, Radio- chemical Centre, Amersham) and 25 ul S100 added at time zero.

If used, actinomycin D (Calbiochem) at a final concentration of 150 ug/ml was added to the RNA synthesis system at time zero.

RNA synthesis was carried out at 3O°C for the lengths of time indicated in Results. The reaction was terminated by

adding 50 ul extraction buffer (10 mM NaCH-jCOO pH 7.0, 1% SDS) containing 50 ug of a mixture of yeast 5S and tRNA (Sigma).The

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RNA was extracted with phenol-chloroform (1/1) at room

temperature and was precipitated from the aqueous phase with 2 volumes of ethanol.

Gel electrophoresis.

RNA was resuspended in 20 ul 10 mM Tris-HCl pH 7.0, 5 M urea and 0.001% xylene blue. The samples were subjected to electrophoresis in polyacrylamide-urea (10% acrylamide-

7 M urea slab gels (19). Unlabeled 5S RNA from X. borealis, a gift from Dr Duri Rungger, was used as marker. RNA was

visualized by staining the gels with ethidium bromide and [ P]32 or [ H] was localized by autoradiography or fluorography (18), respectively. The autoradiographs were scanned with a

Joyce-Loebl Mklllc microdensitometer and the peaks

corresponding to 5S RNA were integrated. Thereafter [ P] or [ H] in 5S RNA was quantitated in excised gel slices by Cerenkov counting or in Instagel (Packard), respectively.

RESULTS

Stimulation of 5S RNA synthesis in polyoma-infected mouse kidney cell cultures.

Synthesis of the polyoma T-antigens became detectable by radioimmune assay 7-9 h after infection. Determined by the immunofluorescence reaction, about 1%, 50-80% and virtually 100% of the nuclei were positive by 12, 16 and 25 h, respectively. Virus-induced stimulation of cellular hnRNA (hnRNP) synthesis became detectable around 12 h, whereas stimulated 45S pre-rRNA, tRNA and 5S RNA synthesis could be detected at the earliest around 16 h after infection (Matter, J.-M.,

Khandjian, E.W. & Weil, R., unpublished results). Determined colorimetrically, polyoma-infected cultures contained by 16 h about 5% and by 25 h about 30% more total RNA and protein than mock-infected controls (2).

To compare synthesis of 5S RNA, polyoma- and mock-infected parallel cultures were pulse-labeled with [ Hjuridine for 1 h at different times between 6 and 30 h after infection; 5S RNA was extracted with phenol from the cytoplasmic fraction and was analysed by electrophoresis in polyacrylamide-urea slab gels.

In mock-infected cultures 5S RNA synthesis remained virtually

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a b a b

5.8 S RNA

5S RNA tRNA

Figure 1

Gel electrophoresis of the small cytoplasmic RNA's from mock- and polyoma- infected mouse kidney cell cultures.

Cultures were labeled with [3H]uridine from 24 to 25 h after infection.Ten ug per slot of cytoplasmic PNA from mock- (lane a) or polyoma-infected (lane b) cultures were analysed by electrophoresis in a polyacrylamide slab gel.

Fluorography was for 5 days.

unchanged throughout the experiments. In polyoma-infected cultures a small but reproducible increase in the apparent rate of 5S RNA synthesis could be detected at the earliest around 16 h after infection (data not shown) whereas by 25 h it was 1.8-2.0 fold higher than in mock-infected controls (figure 1 ) . Determined by A.,,, in polyacrylamide-agarose tubular gels, cytoplasmic extracts from polyoma-infected cultures contained by 25-30 h about 30% more small RNA's (tRNA and 5S RNA) than extracts from mock-infected controls.Little, if any 5S RNA was found in the nuclear fractions (data not shown).

Increased 5S RNA synthesis with S100 from polyoma-infected cultures.

S100 were extracted from polyoma- (SlOO-Py) and mock- infected (S100-M) cultures 15 and 25 h after infection and used to transcribe a cloned'somatic ribosomal 5S gene from

Xenopus borealis (pMB9Xbsl). As expected, synthesis of 5S RNA

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0 10 20 30 U0 minutes Figure 2

Kinetics of 5S RNA synthesis in vitro.

SlOO-Py ( • ) or S100-M ( 0 ) extracted 25 h after infection were incubated with cloned X. borealis 5S genes. The amounts of 5S RNA synthesized were determined after incubation for 5, 10, 20 or 40 min. Lanes a,c,e and g of the insert show the autoradiographs of the products synthesized in SlOO-Py and lanes b,d,f, and h those synthesized in S100-M after incubation for 5,10,20 and 40 min, respectively.

depended on the presence of the plasmid and was inhibited by the addition of 150 ug/ml actinomycin D (12) to the assay mixtures at time zero (data not shown). To determine the

kinetics of 5S RNA synthesis, SlOO-Py and S100-M were incubated for different lengths of time between 5 and 180 min and the amounts of 5S RNA synthesized were quantitated by electrophoresis in slab gels. Experiments with 10 individual preparations of S100 showed that the amounts of 5S RNA increased with time of incubation and reached both in S100-M and SlOO-Py a maximum plateau after 10-30 min which remained unchanged after further incubation for 2 h (figure 2 and unpublished data). In all subsequent experiments we therefore used an incubation time of 60 min. As determined in 10

individual experiments, S100-M and SlOO-Py extracted 15 h after

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p Figure 3

_^_ Detection of polyoma T-antigens by radioinrmune assay. Three mouse kidney cell cultures were labeled with [35s]raethionine (50 uCi/culture) from 15 to 25 h after infection with polyoma

" • *"LT virus. T-antigens in the nuclear fraction (lane A) and in the SlOO-Py (lane B) were isolated by immunoaffinity chromatography, analysed by electrophoresis in a SDS/poly-

«-MT acrylamide (12.5% acrylamide) slab gel and revealed by fluorography (2 weeks exposure).

The positions of large (LT), middle (MT) and small (ST) T-antigens are indicated by arrows.

The other bands are coprecipitated host cell proteins (4).

-ST

front •

infection synthesized essentially the same amounts of 5S RNA (data not shown) whereas SlOO-Py extracted by 25 h synthesized 1.5-2.0 times more 5S RNA than S1OO-M (figure 2 ) . In all

experiments variable amounts of heterogeneous [ P]-labeled RNA formed a broad smear in the gel which was also present after incubation without plasmids. Since this RNA was not observed after pretreatment of S100 with DNAse I it seems to reflect transcription of trace amounts of contaminating mouse cell DNA.

Removal of polyoma T-antigens from SlOO-Py does not alter 5S RNA synthesis.

Mock- and polyoma-infected cultures were labeled with [ S]methionine from 15 to 25 h after infection. S100 and nuclear fractions were assayed for T-antigens by immunoaffinity chromatography, followed by gel electrophoresis.As expected, no T-antigens could be detected in mock-infected cultures.Figure 3 shows that the bulk of large, middle and small T-antigens was present in the SlOO-Py whereas little if any T-antigens could be detected in the nuclear fraction. Aliquots of SlOO-Py and

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A R T Figure 4

M ~ 21 Effect of removal of the polyoma T-antigens on 5S RNA synthesis in vitro. SlOO-Py extracted 25 h after infection was subjected to 3 cycles of immunoprecipitation either with hamster polyoma anti-T serum (lane A) or with normal hamster serum (lane B ) . S100-M was subjected to 3 cycles of imraunoprecipitation with hamster polyoma anti-T serum (lane C).Treatment of an aliquot of S100-M with normal hamster serum gave the same result as in lane C (data not shown). 5S RNA was quantitated by gel electrophoresis.

5S- • *

S100-M were subjected to 3 consecutive cycles of immunoprecipitation with polyoma anti T-serum, a treatment which led to the complete removal of [ S]-labeled T-antigens from SlOO-Py. To other aliquots of SlOO-Py and S100-M the same volume of normal hamster serum or rabbit anti-BSA serum was added instead of the anti-T serum. The results in figure 4, which are representative of 4 individual experiments, show that removal of the T-antigens from SlOO-Py remained without

detectable effect on 5S RNA synthesis.

Correlation between the protein concentration of the S100 and the amounts of 5S RNA synthesized in vitro.

S100-M and SlOO-Py extracted 15 h after infection exhibited virtually the same protein concentration (7.5+_5%

mg/ml) whereas SlOO-Py extracted 25 h after infection contained about 30% more protein (9.7+5% mg/ml) than S100-M. At all times tested virus-coded proteins accounted for less than 0.1% of total protein (2 and unpublished data). To test whether stimulation of 5S RNA synthesis in vitro was related to the increase in protein concentration we diluted SlOO-Py (figure 5)

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100/0 70/30 50/50 25/75

%Sioo-Py/'/. Buffer Figure 5

Effect of dilution of SlOO-Py on 5S RNA synthesis in vitro.

SlOO-Py extracted 25 h after infection was diluted with

transcription buffer as indicated in the figure and was assayed for 5S RNA synthesis.Lanes a,b,c and d of the insert correspond to the dilutions 100/0, 70/30, 50/50 and 25/75, respectively.

and S100-M (data not shown) with transcription buffer. In all experiments a linear relation between the protein concentration in the assay mixtures and the amounts of 5S RNA synthesized was observed. Lowering of the protein concentration of SlOO-Py to that of S100-M reduced the amount of 5S RNA synthesized in SlOO-Py to that synthesized in S100-M. To rule out that small amounts of T-antigen which had not been removed by immunoprecipitation were responsible for the increased 5S RNA synthesis in vitro we used the following approach. In

3 individual experiments the protein concentration of SlOO-Py, extracted 25 h after infection, was diluted with transcription buffer to the protein concentration of S100-M; SlOO-Py and S100-M were then mixed in the ratios indicated in figure 6 and the mixtures assayed for 5S RNA synthesis. In all experiments the amounts of 5S RNA synthesized were essentially independant of the mixing ratio.

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0/100 30/70 60/40 100/0 7. SiQQ-Py/'/.Sioo-M Figure 6

The effect of mixing of SlOO-Py with S100-M on 5S RNA synthesis in vitro.S100 was extracted 25 h after infection.SlOO-Py (9.7 mg/ml) was diluted with transcription buffer to the protein concentration of S100-M (7.5 mg/ml). Immediately thereafter S100-M and SlOO-Py were mixed at the ratios indicated in the figure and assayed for 5S RNA synthesis. Lanes a,b,c and d of the insert correspond to the mixing ratios O/100, 30/70, 60/40 and 100/0, respectively.

DISCUSSION

In polyoma-infected mouse kidney cell cultures the rate of 5S RNA synthesis began to increase around 16 h, i.e. 7-9 h after onset of T-antigen synthesis. The rate of 5S RNA

synthesis reached a maximum plateau around 25 h after infection when it was 1.8-2.0 times higher than in mock-infected parallel cultures. Stimulation of 5S RNA synthesis in vivo thus

coincided in time with the virus-induced increase in total cellular RNA and protein.

The transcription in vitro of cloned Xenopus borealis 5S genes apparently reflected the in vivo situation. S100-M and SlOO-Py extracted 15 h after infection were indistinguishable with respect to protein concentration and 5S RNA synthesis whereas SlOO-Py extracted at 25 h contained about 30% more

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protein and synthesized 1.5-2.0 times more 5S RNA. Complete removal of the polyoma T-antigens from SlOO-Py by three cycles of immunoprecipitation with anti-T serum remained without effect on stimulated 5S RNA synthesis. However, a linear relationship between 5S RNA synthesis in vitro and protein concentration in SlOO-Py and S100-M was noted.

These observations tend to exclude that polyoma T-antigens exert a direct effect on transcription of 5S genes; they are compatible, however, with the assumption that stimulated transcription of mouse and Xenopus borealis 5S genes in vivo and in vitro, respectively, is the result of the virus-induced increase in host cell proteins, such as RNA polymerase III and specific transcription factors, required for transcription of 5S genes (20).

ACKNOWLEDGEMENTS

We thank Professors Robert G. Roeder and Marco Crippa for useful and stimulating discussions and Mr. Otto Jenni for drawing the figures. This work was supported by grant No 3.474.79 from the Swiss National Science Foundation.

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